Abstract Emerging research supports a role for gut microbiota in early-life programming of metabolism and immunity. Although fungi are part of the commensal gut microbiota, particularly during infancy, they have received much less study for their role in health as compared to bacteria. Until recently, fungi were considered to have only pathogenic interactions with humans. However, new studies support that idea that commensal fungi provide protection from disease. Despite this probable importance to public health, we lack fundamental knowledge about the structure and dynamics of gut fungal microbiota (mycobiomes) and how they co-develop with bacterial microbiomes during early life. This has prevented the potential discovery of important mechanisms for interactions between the microbiota and human physiology. For this R21 research, we propose to begin to close this knowledge gap by studying fungal-bacterial microbiome co-development during the first year of life, taking advantage of a large, diverse infant cohort from the Children's Hospital of Philadelphia Care Network (NIH-funded Microbiome, Antibiotics and Growth Infant Cohort [MAGIC] study). In Aim 1, we will gain foundational knowledge about co-maturation of gut mycobiome features with bacterial microbiome structures and functions. This will provide a healthy microbiome benchmark for future studies seeking to understand how microbiomes and interkingdom relationships during early life contribute to later-life health and disease outcomes. To further test the hypothesis that fungal and bacteria microbiomes interact with each other, in Aim 2 we will study the extent to which antibacterial antibiotics disrupt mycobiomes and interkingdom structural and functional relationships, and the durability of these effects. Through these Aims, we expect to gain new knowledge about early-life gut mycobiomes and how they are related to bacterial microbiomes, thereby expanding current microbiome paradigms beyond bacteria to incude fungi and fungal-bacterial inter-kingdom relationships within the human gut . Altogether, these new insights will support further research toward discovering mechanistic links between gut microbiota and human health along the life-span and will provide strong rationale for the development of clincial strategies for the modulation of fungi, along with bacteria, to improve infant and adult health.